Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases

Biochemistry. 2017 Apr 11;56(14):2010-2023. doi: 10.1021/acs.biochem.7b00137. Epub 2017 Mar 31.

Abstract

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αβγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [ Chen , M. , Chou , W. K. W. , Toyomasu , T. , Cane , D. E. , and Christianson , D. W. ( 2016 ) ACS Chem. Biol. 11 , 889 - 899 ]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20 diterpene products and three C25 sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkyl and Aryl Transferases / chemistry*
  • Alkyl and Aryl Transferases / genetics
  • Alkyl and Aryl Transferases / metabolism
  • Aspergillus / enzymology
  • Aspergillus / genetics*
  • Cyclization
  • Diterpenes / metabolism
  • Gene Expression
  • Isomerases / chemistry*
  • Isomerases / genetics
  • Isomerases / metabolism
  • Kinetics
  • Models, Molecular
  • Mutant Chimeric Proteins / chemistry*
  • Mutant Chimeric Proteins / genetics
  • Mutant Chimeric Proteins / metabolism
  • Protein Domains
  • Protein Engineering
  • Protein Structure, Secondary
  • Saccharomycetales / enzymology
  • Saccharomycetales / genetics*
  • Sesterterpenes / biosynthesis
  • Taxus / enzymology
  • Taxus / genetics*

Substances

  • Diterpenes
  • Mutant Chimeric Proteins
  • Sesterterpenes
  • fusicoccadiene
  • Alkyl and Aryl Transferases
  • ophiobolin F synthase, Aspergillus clavatus
  • Isomerases
  • taxa-4(5),11(12)-diene synthase